Action Potential Gating of Calcium Channels and Transmitter Release

The regulation of transmitter release at the neuromuscular junction is tightly regulated by the influx of calcium in the presynaptic nerve terminal. Interestingly, the probability that release sites at the neuromuscular junction will liberate transmitter during each action potential is very low. The reasons for this low probability of release are not well understood. To test the hypothesis that individual N-type calcium channels open with a low probability, single channel recordings of N-type voltage-gated calcium channels were performed. Using this approach I determined the conductance of these channels, their probability of gating during an action potential waveform, and the magnitude of calcium flux during a single channel opening. I conclude from these studies that N-type voltage-gated calcium channels have a very low probability of opening (< 5%) during an action potential and the characteristics of calcium entry during single channel openings can help to explain the low probability of transmitter release at release sites in the neuromuscular junction. To understand how calcium current is activated physiologically, the activation and resulting current from N-type calcium channels elicited by different action potential waveforms were studied. This work was carried out at both room temperature and 37°C to provide a physiological context. Using the whole-cell patch clamp techniques, I studied the activation of current during various action potential shapes and conditions, and the kinetics of N- and L-type current activation. Using this approach I determined that N-type channels activate more slowly than L-type. Furthermore, depending on the action potential shape used and the temperature, action potentials can activate varying proportions (I/Imax) of N-type calcium current (ranging from 10-100%). Under physiological conditions using a frog motoneuron action potential waveform I determined that there was a very low proportion of calcium current activated by a natural action potential (~32%). Adenosine 5´-triphosphate (ATP) is co-released with acetylcholine (ACh) at the neuromuscular junction, and has been found to inhibit transmission. I used the cutaneous pectoris muscle of the Rana pipiens to study ATP-mediated modulation of ACh release. Intracellular postsynaptic recordings were used as a measure of ACh release, and agents that perturb the ATP signaling were examined